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Electromechanical Effects of Protamine and Verapamil in Rat Papillary Muscle

M ah Topcuolu, MD, Mustafa Itein, PhD^1,, Gülay Loolu, MD^2,, smail Günay, PhD^1,, Acar Tokcan, MD, Tümer Ulus, MD

Department of Thoracic and Cardiovascular Surgery 1 Department of Biophysics 2 Department of Physiology Çukurova University Medical Faculty Adana, Turkey

For reprint information contact: M ah Topcuolu, MD Tel: 90 322 338 6627 Fax: 90 322 338 6572 email: sahtopcu{at}pamuk.cc.cu.edu.tr Department of Thoracic and Cardiovascular Surgery, Çukurova University Medical Faculty, Balcali, Adana 01330, Turkey.

	Abstract

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The electromechanical effects of protamine sulfate and the calcium channel blocker verapamil on rat cardiac and skeletal muscles were studied using isolated left ventricular papillary muscle and phrenic nerve-hemidiaphragm preparations. Protamine produced significant decreases in isometric force in the cardiac tissue and contracture developed at concentrations of 40 and 80 mg•L^–1. Isometric force also decreased significantly with verapamil at concentrations of 0.757 and 7.57 mg•L^–1. Both drugs caused significant decreases in the contractile force of hemidiaphragm muscle when the tissue was stimulated indirectly. Protamine and verapamil caused the resting membrane potential and the amplitude of the action potential to decrease in cardiac tissue and overshoot failed to develop with 80 mg•L^–1 of protamine or 7.57 mg•L^–1 of verapamil. These bioelectrical changes developed in a dose-dependent manner. It was concluded that protamine had a similar effect to that of calcium channel blockers and it may act through a reduction of cellular calcium. This effect on cardiac tissue may be mediated through the sarcolemmal ion pumps or channels, leading to changes in calcium homeostasis.

	Introduction

TOP Abstract Introduction Materials and Methods Results Discussion References

 
Protamine sulfate is a polycationic peptide that is used clinically to neutralize the anticoagulant effects of heparin. Systemic hypotension is a frequently observed and unwanted side effect of protamine infusion. Direct vasodilating effects of protamine or its influence on myocardial inotropism have been proposed as an explanation for this hypotension.^1–9 The purpose of this study was to define the direct effects of protamine on action potential characteristics and the contractile function of isolated rat cardiac papillary muscle and to compare these effects with those of a calcium channel blocker, verapamil. To elucidate the underlying mechanisms, the mechanical effects of protamine and verapamil were also studied on rat diaphragm muscle that unlike cardiac muscle, does not require Ca²⁺ entry from extracellular fluid to trigger the release of Ca²⁺ stored in the sarcoplasmic reticulum.

	Materials and Methods

TOP Abstract Introduction Materials and Methods Results Discussion References

 
Fifty male Wistar rats weighing 260 to 300 g were used in this study. Before excision of the heart, the animals were anesthetized with pentobarbital sodium (30 mg•kg^–1, intraperitoneally) and 500 IU heparin per 100 g was administered intravenously before cannulating the trachea. After sternotomy, the pericardium was incised. The ascending aorta was dissected free and cannulated for perfusion on a Langendorff system.¹⁰ The heart was excised, washed 3 times with Krebs buffer to remove heparin, and perfused with oxygenated (equilibrated with 95% O₂ and 5% CO₂) Krebs solution at 30°C to ensure complete removal of heparin. The composition of the Krebs solution was: 113 mmol•L^–1 NaCl; 4.7 mmol•L^–1 KCl; 1.2 mmol•L^–1 MgSO₄; 1.9 mmol•L^–1 CaCl₂; 1.2 mmol•L^–1 KH₂PO₄; 25 mmol•L^–1 NaHCO₃; and 11.5 mmol•L^–1 glucose. After an adaptation period of 15 to 20 minutes to stabilize the tissue to in vitro conditions, left ventricular papillary muscles, including the chordae tendineae, were rapidly isolated. The isolated muscles had a mean (± standard deviation) length of 5.5 ± 0.5 mm, width 1.3 ± 0.2 mm, and weight 31 ± 3 mg. Phrenic nerve-hemidiaphragm preparation was carried out as described by Kelsen and Nochomovits¹¹. Six rats were sacrificed by cervical dislocation and the excised phrenic nerve-hemidiaphragm preparations (mean weight, 53 ± 6 mg; length, 1.8 ± 0.1 cm) were perfused in a tissue bath with oxygenated Krebs solution pH 7.34 to 7.45 at 30°C.

For biomechanical recordings, the papillary muscle was placed between platinum electrodes and suspended in a tissue bath containing 20 mL of oxygenated Krebs solution at 30°C. After an equilibration period of one hour, supramaximal stimulating voltage (15% higher than the maximal pulse required for stimulation of all muscle fibers) and the length at which peak developed tension was maximal were determined. The preparation was equilibrated for 10 minutes until peak developed tension reached a steady state. Supramaximal impulses were delivered at 0.1 Hz as square waves of 0.5 msec duration with a Harvard double-channel stimulator (Harvard Apparatus Ltd., Edenbridge, England, UK). Isometric force of contraction was recorded for 80 minutes on a Grass polygraph (model 7; Grass Instrument Co., Quincy, MA, USA) and a Hitachi digital storage oscilloscope (VC-6045; Hitachi Corp., Tokyo, Japan). In the first series of experiments, measurements were obtained at baseline (recorded for 15 minutes) and then in the presence of protamine over a period of 65 minutes. Protamine sulfate (Sigma, St. Louis, MO, USA) was added to the bath in final concentrations of 20, 40, and 80 mg•L^–1, equivalent to serum concentrations obtained with protamine doses of 1.25, 2.5, and 5 mg•kg^–1 respectively.¹² In the second series of experiments, biomechanical recordings of papillary muscles were made in the presence of the calcium channel blocker verapamil hydrochloride (Knoll AG, Ludwigshafen, Germany) in final concentrations of 0.0151, 0.0757, 0.757, and 7.57 mg•L^–1. The effects of verapamil were examined for 50 minutes after recording baseline values for 10 minutes.

In the first series of experiments on the phrenic nerve-hemidiaphragm preparations, the isometric force of contraction was recorded at baseline after an equilibration period of 15 minutes and at final con-centrations of 20, 40, and 80 mg•L^–1 protamine sulfate. In the second series of experiments, 55.5 mg•L^–1 of verapamil hydrochloride was added to the tissue bath. Direct (via muscle) and indirect (via nerve) stimuli were delivered for 0.5 msec at 0.1 Hz and developed contraction forces were recorded over 30 minutes during verapamil exposure and at each dose of protamine.

For bioelectrical recordings, papillary muscle preparations were placed in a microelectrode tissue chamber perfused with oxygenated Krebs solution at pH 7.34 to 7.45 and 30°C. The preparations were stabilized in the solution for one hour before measurement. Transmembrane potentials were recorded by means of glass microelectrodes filled with 3 M KCl and connected to a microelectrode amplifier (Nihon Kohden MEZ-7200; Nihon Kohden Corp., Tokyo, Japan). Electrical events were displayed on a digital storage oscilloscope (Hitachi VC-6045; Hitachi Corp., Tokyo, Japan) and also recorded. Measurements were made of resting membrane potential, action potential amplitude, overshoot, and action potential duration at 50% repolarization (APD₅₀), at baseline and then at protamine sulfate concentrations of 20, 40, or 80 mg•L^–1. In the second series of experiments, recordings were obtained at verapamil hydrochloride concentrations of 0.0151, 0.0757, 0.757, and 7.57 mg•L^–1.

Values were expressed as the mean ± standard deviation. Statistical analysis was performed using paired t tests. Values of p less than 0.05 were regarded as being statistically significant.

	Results

TOP Abstract Introduction Materials and Methods Results Discussion References

 
In isolated rat papillary muscle, administration of protamine to the bathing solution produced significant decreases in isometric force in a dose-dependent manner (p < 0.001) as detailed in Table 1. After protamine was completely washed off the papillary muscle (by washing with Krebs solution 5 or 6 times), it failed to contract when an electrical stimulus was delivered, indicating that the effect of protamine was irreversible. At protamine sulfate concentrations of 40 and 80 mg•L^–1, contracture developed in the papillary muscle (Figure 1). Isometric force also decreased significantly at verapamil hydrochloride concentrations of 0.757 and 7.57 mg•L^–1 (Table 2).

View larger version (38K): [in this window] [in a new window]   Figure 1. Trace showing contracture development in a rat papillary muscle at protamine concentrations of 40 and 80 mg•L–1.

	Discussion

TOP Abstract Introduction Materials and Methods Results Discussion References

In the presence of verapamil, electromechanical changes in the cardiac papillary tissue were similar to those observed with protamine administration, except for APD₅₀ that was significantly prolonged in the presence of 80 mg•L^–1 protamine compared with baseline values. Lin and colleagues⁶ determined that in addition to negative inotropy, 10 to 30 mg% protamine was able to decrease the amplitude of the oscillatory afterpotentials and aftercontractions induced by cardiotonic drugs. Therefore, protamine may depress the activity in rat papillary fibers through a reduction of cellular calcium or the oscillatory inward currents during diastole, similar to the action of calcium channel blockers.⁶ Our results suggest that protamine may inhibit the cardiac sarcolemmal ionic pump, causing the observed changes in resting membrane potential and action potential characteristics.⁹ The depolarization induced by protamine may be due to a decrease in K⁺ conductance, since it has been reported that depolarization was in part reversed by increasing the external K⁺ concentration.⁶ It has also been hypothesized that protamine causes a decline in the force of contraction by the development of slow-response action potentials, occurring as a consequence of a decline in membrane K⁺ and Na⁺ conductance, perhaps due to the surface-charge effects of protamine on membranes altering the configuration of ion channels.⁶ Park and colleagues¹² have also shown that protamine caused partial depolarization and reduced the action potential amplitude in ventricular papillary muscle. Protamine was shown to irreversibly decrease the inward current in K⁺ and Ca²⁺ channels.¹²

Similar to the findings in this study, high concentrations of protamine were noted to cause a dose-dependent depression of contractile force and to induce contracture in feline papillary muscle.^13,14 This provides evidence that protamine also affects myocyte active relaxation processes (Table 1). Further, Wakefield and colleagues¹⁵ reported that protamine decreased adenosine triphosphate production in endothelial cells. Thus, the alterations in myocyte active relaxation with protamine may be due to reduced adenosine triphosphate and diminished Ca²⁺-adenosine triphosphatase activity.

When the effects of verapamil and protamine on the contractile force of skeletal muscle (hemidiaphragm preparation) were evaluated, both drugs were observed to be ineffective during direct stimulation but they were effective when the muscle was stimulated indirectly via the phrenic nerve (Table 3). This suggests that protamine, similar to calcium channel blockers, may act through a reduction of cellular calcium that must increase to cause presynaptic release of the neurotransmitter acetylcholine.

In view of the observed effects of protamine on the electromechanical properties of cardiac and skeletal muscles, it is possible to conclude that protamine may influence the function of sarcolemmal-based ion pumps or channels, leading to changes in Ca²⁺ homeostasis and that this may cause fundamental changes in the myofilament contractile apparatus. Further studies investigating the mechanisms by which protamine depresses myocyte contractile function are warranted.

	References

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Alert me to new issues of the journal Add to Personal Folders Download to citation manager Permission Requests Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Topcuolu, M S. Articles by Ulus, T. Search for Related Content PubMed Articles by Topcuolu, M S. Articles by Ulus, T.